Walrant Astrid, Saxton Daniel S, Correia Guilherme Pereira, Gallop Jennifer L
Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Biochemistry, University of Cambridge, Cambridge, UK.
Methods Cell Biol. 2015;128:125-47. doi: 10.1016/bs.mcb.2015.01.020. Epub 2015 Apr 8.
Xenopus egg extracts are a powerful tool to reconstitute complex cell biological processes using a cell-free strategy. When used in conjunction with liposomes and supported lipid bilayers, they can recapitulate the biochemical activities occurring at the cytosol/plasma membrane interface of the cell that underlie remodeling of the actin cytoskeleton. We use these in vitro systems to elucidate how membranes and proteins collaborate to make the appropriate actin structure at a given time and place. We have recently broadened the types of membrane substrate used, and also optimized protocols for preparation of Xenopus egg extracts for actin assembly assays from membranes. Tuning the lipid composition and curvature appropriately demands an appreciation of the native phospholipid and curvature environments that can form transiently in cells. Supported lipid bilayers on glass coverslips that contain phosphatidylserine and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) make actin bundles termed filopodia-like structures that contain fascin and have vasodilator-stimulated phosphoprotein (VASP) at their growing tips, indicating that these resemble filopodia growing from the plasma membrane. The combination of PI(4,5)P2 and phosphatidylinositol 3-phosphate in curved liposomes or supported bilayers on glass nanospheres uses Snx9, Cdc42, N-WASP (neuronal-Wiskott-Aldrich syndrome protein), and Arp2/3 complex for actin polymerization suggesting that this membrane may mimic the progression from plasma membrane to endosomes. Here we describe how to prepare high-speed supernatant frog egg extracts and phosphoinositide-containing liposomes and supported lipid bilayers that can assemble actin structures. We also describe the methods we use to assay actin polymerization using microscopy and spectrofluorometry and our protocol for immunodepleting specific proteins from extracts.
非洲爪蟾卵提取物是一种强大的工具，可利用无细胞策略重建复杂的细胞生物学过程。当与脂质体和支撑脂质双层结合使用时，它们可以重现细胞胞质溶胶/质膜界面发生的生化活动，这些活动是肌动蛋白细胞骨架重塑的基础。我们使用这些体外系统来阐明膜和蛋白质如何协同作用，在特定的时间和地点形成合适的肌动蛋白结构。最近，我们拓宽了所使用的膜底物类型，并优化了用于从膜进行肌动蛋白组装测定的非洲爪蟾卵提取物的制备方案。适当地调整脂质组成和曲率需要了解细胞中可能瞬时形成的天然磷脂和曲率环境。含有磷脂酰丝氨酸和磷脂酰肌醇4,5-二磷酸（PI(4,5)P2）的玻璃盖玻片上的支撑脂质双层会形成称为丝状伪足样结构的肌动蛋白束，这些结构含有成束蛋白，并且在其生长尖端具有血管舒张刺激磷蛋白（VASP），这表明它们类似于从质膜生长的丝状伪足。弯曲脂质体或玻璃纳米球上的支撑双层中PI(4,5)P2和磷脂酰肌醇3-磷酸的组合使用Snx9、Cdc42、N-WASP（神经元Wiskott-Aldrich综合征蛋白）和Arp2/3复合物进行肌动蛋白聚合，这表明这种膜可能模拟了从质膜到内体的进程。在这里，我们描述了如何制备可组装肌动蛋白结构的高速上清蛙卵提取物和含磷酸肌醇的脂质体及支撑脂质双层。我们还描述了我们使用显微镜和荧光光谱法测定肌动蛋白聚合的方法，以及我们从提取物中免疫去除特定蛋白质的方案。
